| Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV) is a severe infectious disease in swine herds characterized by reproductive failure in sows and respiratory illness in young pigs. It was first described in the United States in 1987 and a few years later in European, and in China in 1995. PRRS has been recognized as one of the most economically important diseases in the swine industry worldwide.1 PRRSV strain was isolated from the clinical tissue samples from farms in Henan province, the isolate was designated as PRRSV HN-08. The biological characteristics of the isolate shows that the virus partical with diameter of 50nm is orbicular and is RNA virus, it can reproduce in Marc-145 cells and can produce CPE, may combine with PRRSV fluorescently-labeled antibody, neutralize by PRRSV positive serum, and it is sensitive to heat, acid, alkali and chloroform. Pathogenicity of porcine tests showed that the virulence of HN-08 is stronger than earlier PRRSV isolates. The antigen monitoring results show that propagation speed of the mutant PRRSV is quicker in body, duration of viremia is longer, distribution in the apparatus is wider.To more fully understand the genetic diversity of PRRSV isolates, we analyzed and compared the GP5 and Nsp2 sequences of PRRSVs which were isolated from 2005 to 2008 in mainland of China. GP5, the most important immunogenicity and neutralizing antigen of PRRSV, has the highest genetic diversity under the pressure of environment and immunity. 37 GP5 sequences collected from the clinical samples by our lab were compared with the 14 sequences in GenBank. As a result, all the Chinese isolates examined belong to the NA-type. The identity of 85.6%-99.5% was observed among PRRSV isolates, which shows genetic diversity of ORF5 is high with an annual feature and no obvious geographical characteristics. L39 mutant I39 in the antibody binding site of the primary neutralizing epitope of the 29 high pathogenic PRRSV isolates. Two key amino acids, which were considered to be involved in PRRSV attenuation, were accordant with the VR-2332 strain, CH-1a strain. Decoy epitopes are conservative among PRRS iaolates. Nsp2 protein is highly antigenic. 8 strains isolated in 2005 have no deletion in the Nsp2 protein, while a deletion of 30 amino acids was found in the Nsp2 protein of 29 strains isolated from 2006 to 2008. Phylogenetic analysis and amino acid homogeneity showed that genetic diversity of Nsp2 is high but the deletion of 30 amino acids maybe not involved into the reproduction of PRRSV. The relationship between PRRSV isolates and the RespPRRS/Repro vaccine strain is far.In the present study, the gene of PRRSV HN-08 strain was sequenced and analized. Phylogenetic and amino acid homogeneity analysis showed that the high pathogenic PRRSV isolates share the same origin.In order to manipulate the PRRSV in vitro and study for gene function and replication of the virus, the recombined plasmid including the full length genomic cDNA clone of PRRSV was constructed.4 cDNA fragments covering the PRRSV genome were amplified. After several cycles of cloning and subcloning, a full-length genomic cDNA clone of PRRSV was obtained. Pâ…¡, Pâ… and HamRz were introduced immediately upstream of 5'-NTR, HdvRz, Tâ… and Tâ…¡was introduced into the cDNA clone downstream of 3'-NTR.There are some mutants in the recombinant plasmids, but no terminal mutation were found after sequencing. The recombined plasmids pPâ… T7Pâ…¡-HN08 including the complete gene of PRRSV HN-08 was obtained. The rescued virus was not acquired after pPâ… T7Pâ…¡-HN08 was transfected into Marc-145.The construction of a full-length genomic cDNA clone of PRRSV is a crucial step to obtain the infectious clone. Further study will be going to test infectivity of the cDNA clone and look insight the structure and function of PRRSV genome.
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